Summary This request for a supplement to R01DK115380 is for funds for additional acquisition of magnetic resonance spectroscopy measures of choline content of liver in humans. The parent study seeks to develop a better biomarker panel that can be used by clinicians to assess a person's choline status. It has become apparent to us during the pilot phase of our studies that assessing Cho pool size using isotope dilution needs to be complemented by another accepted measure against which we can validate our metabolomic biomarker panel. We believe that using magnetic resonance spectroscopy to measure in vivo pools of soluble choline metabolites in liver needs to be added to our studies if we are to validate the metabolomic biomarker panel that we develop so as to convince clinicians that our metabolomic biomarker panel reliably predicts choline status. Choline is an important dietary supplement and essential nutrient, but there are no good validated biomarkers for assessing choline nutritional status that can be practically applied in clinical or public health practice. Choline is a required nutrient with Adequate Intake (AI) and a Tolerable Upper Limit (UL) levels set by the U.S. Institute of Medicine's Food and Nutrition Board and a Recommended Daily Intake (RDI). Given the narrow range for healthy intake of choline, given the establishment of a RDI of choline to maintain health, given the 3-fold variation in dietary intake in the US, given the effects of common genetic variants on requirements for choline and because plasma choline concentrations do not adequately reflect status, we propose that a panel of laboratory tests be developed and validated that assess choline status in humans. We will identify the biomarkers that best correlate with measurements of choline pool size using isotope dilution. Healthy volunteers (n=50 males, 50 premenopausal females and 50 postmenopausal females), as outpatients, will consume meals containing 100% of the RDI of choline (550 mg) for 2 weeks, 50% (275 mg) of the RDI of choline for 2 weeks, and 25% (137.5 mg) of the RDI of choline for 2 weeks. The order of dietary periods is randomly assigned and subjects and staff are masked. All subjects will receive all diets while on study, with a minimum of 2 weeks of ?washout? between diet periods where participants go back to their normal diet. On Day 12 of each diet period, fasted subjects will consume a 250 mg bolus of choline in the form of Cho chloride-(trimethyl-d9). Plasma and urine samples will be collected for metabolomic assays and isotope dilution estimation of choline pool size and we will perform transient elastography of liver to assess liver fat. Magnetic resonance will also be used for independent assessment of choline status and liver fat. These studies will be used to determine the optimal set of biomarkers to use in calculating a choline status score.